Fluorescence Lifetime for On-the-Fly Multiplex Detection of DNA Restriction Fragments in Capillary Electrophoresis

• Published in: Analytical chemistry (Washington) 2000-11, Vol.72 (21), p.5444-5449
• Main Authors: McIntosh, Sara L, Nunnally, Brian K, Nesbit, Ashlyn R, Deligeorgiev, Todor G, Gadjev, Nikolai I, McGown, Linda B
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; capillary electrophoresis; Chemistry; Deoxyribonucleic acid; DNA; DNA - chemistry; Dna, deoxyribonucleoproteins; Electrophoresis, Capillary; Fluorescence; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; Indicators and Reagents; Intercalating Agents; Nucleic acids; Spectrophotometry, Ultraviolet
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac000235t

This paper describes the first use of frequency-domain fluorescence lifetime for multiplex detection of DNA restriction fragments in capillary electrophoresis (CE). The fragments were labeled with monomeric intercalating dyes that can be excited by either the 488- or 514-nm line of an argon ion laser and have lifetimes in the range of 0.5−2.5 ns. We were able to achieve multiplex lifetime detection in the CE separation of a restriction fragment digest and a DNA size ladder in the same run, for fragments shorter than 700 bp. Different gel buffer systems, including a modified polyacrylamide gel and several tris−borate−EDTA/hydroxyethylcellulose (TBE/HEC) gels, were investigated for separation and detection of the dye-labeled DNA fragments. Best results for both electrophoretic resolution and lifetime detection were obtained using a gel containing 1% high molecular weight (90 000−105 000) HEC and 0.3% low molecular weight (24 000−27 000) HEC in TBE buffer.